SIRT5 Protein, Human (Flag)

Materials
Assay buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0
TCNB buffer：50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
PRSS3/Trypsin-3 Protein, Human (HEK293, His) (HY-P75985)
Activator Protein: Enterokinase Protein, Bovine (His)
Stop Solution: 8 ng/μL Trypsin, 4 mM Nicotinamide, 50 mM Tris, 100 mM NaCl, 30% (v/v) Isopropanol, pH 8.0
Beta-NAD: Dilute in ultrapure water to 100 mM
Substrate: FLUOR DE LYS?-Succinyl, Desuccinylase Substrate (5 mM)
Standard: 7-Amino-4-methylcoumarin (HY-D0027)

Procedure
1. Prepare a standard curve using AMC standards with concentrations of 10000, 5000, 2500, 1250, 625, 312.5, 156.25, 78.125, 19.53, 9.76, 0 pmol. Read the fluorescence at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
2. Activation of Human Trypsin-3:
a. Dilute Bovine Enterokinase to 2 μg/mL in TCNB buffer;
b. Dilute Human Trypsin-3 to 200 μg/mL in TCNB buffer;
c. Mix equal volumes of the diluted proteins;
d. Incubate overnight at room temperature.
3. Dilute the Human SIRT5 to 10 μg/mL in assay buffer.
4. Dilute the substrate to 100 μM in assay buffer containing 2 mM beta-NAD.
5. Add 25 μL of 10 μg/mL Human SIRT5 and 25 μL of diluted substrate to the wells. For the control group, add only 25 μL of 10 μg/mL Human SIRT5.
6. Seal the plate and incubate at 37°C for 30 minutes.
7. Add 50 μL of stop solution to all wells and mix. For the control wells, add the stop solution first and then add 25 μL of substrate.
8. Seal the plate and incubate at room temperature for 15 minutes.
9. Read the fluorescence in endpoint mode at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
10. Calculate specific activity: